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Immunoassays for tropical parasitic infections
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICALMEDICINEAND HYGIENE (1993) 87, 497-498
lmmunoassays
for tropical
parasitic
497
infections
Alister Voller Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK Introduction
Over the last decadethere has been an explosion of interest in and development of immunodiagnostic tests for infectious diseases.The technical developments are most easily considered in terms of reagents and of methods. The tremendous advances in biotechnology have meant that the biggest changes have been in the reagents, namely, antibodies and antigens used. Monoclonal antibodies have been raised to virtually every antigen of all the parasitic protozoa. These antibodies are highly specific and can provide the basis of much improved tests for antigen detection. Monoclonal antibodies are now used routinely as the raw material in immunofluorescent tests (e.g., to demonstrate Entamoeba) and in enzymelinked immunosorbent assays(ELISAS) (e.g., for malaria antigen detection); they have the great advantage that large amounts of defined, standardized reagents can be produced. Monoclonal antibody-based ELISAS or dot-blot systemshave proved most valuable for the demonstration of protozoa1 parasites, or their antigens, in their vertebrate hosts, vectors and the environment. One result of the recent emphasis on vaccine development has been a much improved understanding of the make-up of parasites, and this is having a significant impact on immunodiagnostics. The analysis of the structure of the major antigens has led to the identification of those specific antigens best suited for use in tests to detect the host’s antibodv response. Such antigens can be nrenared on a large scale by-peptide synthesrs or by recombinant technoloav and can markedlv imnrove the snecificitv of tests for Gtibody (e.g. ELI& for *Trypanosom> cruzi ahtibody). The developments in methods have been less dramatic than those in reagents in recent years, and consist mainly of improvement and consolidation of existing methods rather than truly innovative approaches. Those techniques which employ a ‘labelled’ reagent dominate the field at present. One of these, immunofluorescence (or fluorescent antibody) was for long the test of choice but it has now largely been replaced by ELISA which, using enzymes as labels, is more suitable for large scaleuse and which yields objective results. ELISA has the added advantage of being useful for both antigen and antibody detection. The various dot-blot tests are essentially similar but use antigens on paper or plastic supports to detect antibody which is then visualized using antiglobulins labelled with enzymes, dyes or colloidal gold. These dotblot methods are more convenient, especially as ‘dipstick’ techniques, even though they may be less suitable for large scale screening applications. The PATH@ HIV dipstick is an example of a dot system using colloidal gold and is easy and cheap to use, and gives rapid reliable results (KERCKHOVEN et al., 1991). It is seen by some as the model for other test systemsof the future. Simple agglutination tests are attractive propositions and would be ideal for use in the tropics if they could be made reliable. Those dependent on antigen/antibody coated ervthrocytes are -invariably troublesome and give substantial numbers of false positive reactions in the tronics. This is one reason why ihey have gone out of fashion for hepatitis B surface antigen (HBsAg) testing. However, a new agglutination test for human immunodeficiency virus (HIV) based on antigen coupled to a monoclonal antibody against human red blood cells is more suitable for tropical use, since it uses the patient’s own red blood cells as the indicator (RYLATT et al., 1990). Again, this
test format could be extended to other infections. Other agglutination tests using parasites or fragments have also come into prominence for trypanosomiasis and leishmaniasis and are discussedbelow. The real ‘state of the art’ immunoassays are the pregnancy tests currently available. These have reached a new level of user-friendliness and are based on devices incorporating monoclonal antibody-coated beads which, in the presence of human chorionic gonadotrophin, will produce a coloured reaction product. The devices require no measurement of sample or timing, and medical or laboratory training is not required to use them. Unfortunately their cost atpresent puts them beyond the reach of all but the wealthiest nations. Similar devices have been introduced for Chlamydia and for HIV. Until recently, most immune tests were done on serum. This is a rather less attractive proposition nowadays becauseof the hazards associatedwith blood collection and attempts have been made to use alternative noninvasive sampling. Urine is the well established choice for pregnancy tests and has been used for the detection of antigens in schistosomiasis and Chagas disease. Possibly of more general application is antibodv detection in saliva and urine. With properly designed capture ELISAS these give results as crood as serum in an HIV antibodv test ~MORTIMER &-PARRY! 1991) and, presumably, they could be used for detectmg other infections. Applications of immunoassays protozoa1 infections
to tropical
parasitic
Malaria is still the biggest killer diseasein this group and has proved to be a magnet for laboratory workers in the, so far, unrewarding search for a malaria vaccine. A by-product of this work has been the better understanding of the antigenic make-up of the various stagesof the parasite. Monoclonal antibodies to individual antigens have led to the efficient detection of sporozoites in mosquitoes and of Plasmodium falciparum asexual stagesin blood (DUBARRY et al., 1990; TAYLOR & VOLLER, 1993). User-friendly ELISAS for malarial antibody have also been developed, based both on parasitic extracts and recombinant antigens (SRIVASTAVA et al., 1989). These tests could provide the basis for blood transfusion screening but as yet the simple stained blood smear is the only reliable method for diagnosis of acute malaria in individuals, for which purpose it is unchallenged in terms of convenience and cost. Alternatives under development include fluorescent staining (HUNT COOKE et al., 1992) and detection of parasite-specific enzymes(MAKLER & HINRICHS, 1993). For African trypanosomiasis, the immunotests have had a real impact in the form of simple card agglutination tests (CATTS). In these tests an antigen-coated latex particle suspension is simply mixed with the patient’s serum on a plastic card and the agglutination pattern is observed. Virtually all patients with Ttypanosoma brucei gambiense, and about half of those with T. b. rhodesiense, infections are detected by this test (BUSCHER et al., 19911 NOIREAU et al., 1991). which is entirelv adeauate for screening. A monoclonal antibody-based E~ISA for antigen also appearspromising (NANTULYA et al., 1992). Visceral leishmaniasis can also prove difficult to diagnose without invasive procedures and here antibody testing has proved useful both for individual diagnosis and for population screening. ELISXs have been widely used but more recently the direct agglutination test (DAT) has
498
been shown to be as sensitive and specific but easier to oerform (EL SAFI 8~E~~~s.1989). Chagas disease has always attracted the immunologists because the demonstration of antibody is virtually the only practicable way of screening for the infection on a large scale. Immunofluorescence (IFA) has been long established but disadvantages include its lack of objectivity and the labour-intensiveness. ELBA has now largely replaced IFA but still cross reactions with T. rangeli and Leishmania spp. have been a problem. This has now been overcome by the latest ELISAS(KRIEGERet al., 1992), which are based on peptide antigens exhibiting no crossreaction. These ELISASare particularly suited to blood bank screening, where the HIV and HBsAg ELISASin the same format are already well established. For the diagnosis of invasive amoebiasis, tests are carried out to detect antibody to E. histolytica. ELISA has been the method of choice (TAKEUCHI et al.. 1988). in either the microplate or dot-blot format (KAN~AR &‘VINAYAK, 1991). It is likely to be further enhanced when the recombinant antigens presently under evaluation (STANLEYet al., 1991) are introduced. The detection of E. histolytica in faeces has been more of a problem. ELISAS and dot-blot tests have had limited success but recent developments, including monoclonal antibodies which can distinguish between Dathogenic and nonpathogenic strain< offer more promise -(WONSIT et al., ~~~~:GONZALEZ-RUIZetal.. 1993). C&rdiasis is widely distributed in developing countries and the diagnosis is troublesome since it depends on microscopical examination of multiple faecal samples. ELISAS have now been developed to the point where they are sensitive and specific and they are especially well suited to enidemiological studies (GOLDIN et al., 1990). In general,‘antibody detection is of little use in giardiasis; however the demonstration of immunoglobulin M specific antibody may be useful (SULLIVANet al., 1991). It can be seen that there have recently been significant advances in the application of immunological research to the diagnostic problems with tropical parasitic diseases, and I confidently expect that, together with the new diagnostic methods develoDed bv molecular biologists (to be yeviewed in the next issue of the Transaction& these efforts will vield nractical and cost-effective benefits in the shape of’ more widely available user-friendly kits in the near future. I
Acknowledgements
I thank the Overseas Development Administration for their sup ort of the Appropriate Health Technology Programme at the E ondon School of Hygiene and Tropical Medicine.
References
Buscher, I’., Draelawts, E., Magnus, E., Vervoort, T. & Van Meirvenne, N. (1991). An experimental latex agglutination test for antibody to human African trypanosomiasis. Annales de la Societe Belge de Medecine Tropicale, 71,267-273.
Dubarry, M., Luilier, M., Malot, N., Bayard, I’., Lambin, I’., Prou, P. & Monjour, L. (1990). Enzyme immunoassays for detection of malarial antigens in human plasmas by Plasmodium falciparum monoclonal antibodies. American Journal of Tropical Medicine and Hygiene, 43,116-123.
El Safi, S. H. & Evans, D. A. (1989). A comparison of the direct agglutination test and enzyme-linked irnmunosorbent assay in the sero-diagnosis of visceral leishmaniasis in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,334-337.
Goldin, A. J., Apt, W., Aguilera, X., Zulantay, I., Warhurst, D. C. & Miles, M. A. (1990). Efficient diagnosis of giardiasis among nursery and primary school children in Santiago, Chile
by capture ELBA for the detection of fecal Giardia antigens. American Journal of Tropical Medicine and Hygiene, 42, 538-
545. Gonzalez-Ruiz, A., Haque, R., Rehman, T., Aguirre, A., Jaramillo, C., Castanon, G., Hall, A., Ruiz-Palacios, G., Warhurst, D. & Miles, M. A. (1993). A monoclonal antibody for distinction of invasive and non-invasive clinical isolates of Entamoeba histolytica. Journal of Clinical Microbiology, 30, 28072813. Hunt Cooke, A., Moody, A. H., Lemon, K., Chiodini, I’. L. & Horton, J. (1992). Use of the fluorochrome benzothiocarboxypurine m malaria diagnosis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86,378.
Kanwar, J. R. & Vinayak, V. K. (1991). A comparative efficacy of plate ELBA and dot ELBA to detect antiamoebic antibodies in clinical patients. Tropical and Geographical Medicine, 43, 261-265. Kerckhoven, V., Vercauteren, G., Piot, I’. & van der Groen, G. (1991). Comparative evaluation of 36 commercial assays for detecting antibodies to HIV. Bulletin of the World Health Organizati& 69,753-760. Krieger, M. A., Almeida, E., Oelemann, W., Lafaille, J. J., Pereira, J. B., Krieger, H., Carvalho, M. R. & Goldenberg, S. (1992). Use of recombinant antigens for the accurate diagnosis of Chagas’ disease.AmericanJournal of Tropical Medicine and Hygiene, 46,427-434.
Makler, M. T. & Hinrichs, D. J. (1993). Measurement of the lactic dehydrogenase activity of Plasmodium falciparum as an assessmentof parasitaemia. American Journal of Tropical Medicine and Hygiene, 48,205-210.
Mortimer, I’. I’. & Parry, J. V. (1991). Non invasive virological diagnosis: are saliva and urine substrates adeauate substitutes forblood? Reviews in Medical Virology, 1,73-78. Nantulva. V. M.. Doua. F. & Molisho. S. (1992). Diaenosis of Tryp&soma brucei gambiense sleeping ~sickness ising an antigen detection ELISA. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86,42-45.
Noireau, F., Force-Barge, I’. & Cattand, I’. (1991). Evaluation of Testryp CATT applied to samples of dried blood for the diagnosis of sleeping sickness. Bulletin of the World Health Organization, 69,607-608.
Rylatt, D. B., Kemp, B. E., Bundesen, G., John, M. A., O’Reilly, E. J., Cottis, L. E., Mites, S. J., Shaw, J. M., Dmh, D. I’., Stapleton, D. & Hillyard, C. J. (1990). A rapid whole Fp71immunoassay system. MedicalJournal ofAustralia, 152, Srivastava, I. K., Takacs, B., Caspers, I’., Certa, U., McGregor, I. A., Scaife, J. & Perrin, L. H. (1989). Recombinant polypeptides for serology of malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,3 17-32 1. Stanley, S. L., Jackson, T. F. H. G., Read, S. L., Calderon, J., Kunz-lenkins. C.. Gathiram. V. & Li. E. 11991). Serodiaanosis of invasive amoebiasis using a recombinant Entamoebh histolytica protein. Journal of the American Medical Association, 266,1984-1986. Sullivan, I’. B., Neale, G., Cevallos, A. M. & Farthing, M. J. G. (1991). Evaluation of specific serum anti-Giardia IgM antibody response in diagnosis of giardiasis in children. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 748-749. Takeuchi,, T., Matsuda, H., Okuzawa, E., Nozaki, T., Kobayashi, S. & Tanaka, H. (1988). Application of an ELISA to detect anti amoebic antibody in various forms of amoebic infection. JapaneseJournal of Experimental Medicine, 58, 229232. Taylor, D. W. & Voller, A. (1993). The development and validation of a simple antigen detection ELISA for Plasmodium falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87,29-3 1.
Wonsit, R., Thammapalerd, N., Tharavanij, S., Radomyos, I’. & Bunnag, D. (1992). Enzyme-linked immunosorbent assay based on monoclonal and oolvclonal antibodies for the detection of Encamoeba histolyiica antigens in faecal specimens. Transactions of the Royal Society of Tropical Medicine and Hy-
giene,86, 166-169.
lmmunoassays
for tropical
parasitic
497
infections
Alister Voller Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK Introduction
Over the last decadethere has been an explosion of interest in and development of immunodiagnostic tests for infectious diseases.The technical developments are most easily considered in terms of reagents and of methods. The tremendous advances in biotechnology have meant that the biggest changes have been in the reagents, namely, antibodies and antigens used. Monoclonal antibodies have been raised to virtually every antigen of all the parasitic protozoa. These antibodies are highly specific and can provide the basis of much improved tests for antigen detection. Monoclonal antibodies are now used routinely as the raw material in immunofluorescent tests (e.g., to demonstrate Entamoeba) and in enzymelinked immunosorbent assays(ELISAS) (e.g., for malaria antigen detection); they have the great advantage that large amounts of defined, standardized reagents can be produced. Monoclonal antibody-based ELISAS or dot-blot systemshave proved most valuable for the demonstration of protozoa1 parasites, or their antigens, in their vertebrate hosts, vectors and the environment. One result of the recent emphasis on vaccine development has been a much improved understanding of the make-up of parasites, and this is having a significant impact on immunodiagnostics. The analysis of the structure of the major antigens has led to the identification of those specific antigens best suited for use in tests to detect the host’s antibodv response. Such antigens can be nrenared on a large scale by-peptide synthesrs or by recombinant technoloav and can markedlv imnrove the snecificitv of tests for Gtibody (e.g. ELI& for *Trypanosom> cruzi ahtibody). The developments in methods have been less dramatic than those in reagents in recent years, and consist mainly of improvement and consolidation of existing methods rather than truly innovative approaches. Those techniques which employ a ‘labelled’ reagent dominate the field at present. One of these, immunofluorescence (or fluorescent antibody) was for long the test of choice but it has now largely been replaced by ELISA which, using enzymes as labels, is more suitable for large scaleuse and which yields objective results. ELISA has the added advantage of being useful for both antigen and antibody detection. The various dot-blot tests are essentially similar but use antigens on paper or plastic supports to detect antibody which is then visualized using antiglobulins labelled with enzymes, dyes or colloidal gold. These dotblot methods are more convenient, especially as ‘dipstick’ techniques, even though they may be less suitable for large scale screening applications. The PATH@ HIV dipstick is an example of a dot system using colloidal gold and is easy and cheap to use, and gives rapid reliable results (KERCKHOVEN et al., 1991). It is seen by some as the model for other test systemsof the future. Simple agglutination tests are attractive propositions and would be ideal for use in the tropics if they could be made reliable. Those dependent on antigen/antibody coated ervthrocytes are -invariably troublesome and give substantial numbers of false positive reactions in the tronics. This is one reason why ihey have gone out of fashion for hepatitis B surface antigen (HBsAg) testing. However, a new agglutination test for human immunodeficiency virus (HIV) based on antigen coupled to a monoclonal antibody against human red blood cells is more suitable for tropical use, since it uses the patient’s own red blood cells as the indicator (RYLATT et al., 1990). Again, this
test format could be extended to other infections. Other agglutination tests using parasites or fragments have also come into prominence for trypanosomiasis and leishmaniasis and are discussedbelow. The real ‘state of the art’ immunoassays are the pregnancy tests currently available. These have reached a new level of user-friendliness and are based on devices incorporating monoclonal antibody-coated beads which, in the presence of human chorionic gonadotrophin, will produce a coloured reaction product. The devices require no measurement of sample or timing, and medical or laboratory training is not required to use them. Unfortunately their cost atpresent puts them beyond the reach of all but the wealthiest nations. Similar devices have been introduced for Chlamydia and for HIV. Until recently, most immune tests were done on serum. This is a rather less attractive proposition nowadays becauseof the hazards associatedwith blood collection and attempts have been made to use alternative noninvasive sampling. Urine is the well established choice for pregnancy tests and has been used for the detection of antigens in schistosomiasis and Chagas disease. Possibly of more general application is antibodv detection in saliva and urine. With properly designed capture ELISAS these give results as crood as serum in an HIV antibodv test ~MORTIMER &-PARRY! 1991) and, presumably, they could be used for detectmg other infections. Applications of immunoassays protozoa1 infections
to tropical
parasitic
Malaria is still the biggest killer diseasein this group and has proved to be a magnet for laboratory workers in the, so far, unrewarding search for a malaria vaccine. A by-product of this work has been the better understanding of the antigenic make-up of the various stagesof the parasite. Monoclonal antibodies to individual antigens have led to the efficient detection of sporozoites in mosquitoes and of Plasmodium falciparum asexual stagesin blood (DUBARRY et al., 1990; TAYLOR & VOLLER, 1993). User-friendly ELISAS for malarial antibody have also been developed, based both on parasitic extracts and recombinant antigens (SRIVASTAVA et al., 1989). These tests could provide the basis for blood transfusion screening but as yet the simple stained blood smear is the only reliable method for diagnosis of acute malaria in individuals, for which purpose it is unchallenged in terms of convenience and cost. Alternatives under development include fluorescent staining (HUNT COOKE et al., 1992) and detection of parasite-specific enzymes(MAKLER & HINRICHS, 1993). For African trypanosomiasis, the immunotests have had a real impact in the form of simple card agglutination tests (CATTS). In these tests an antigen-coated latex particle suspension is simply mixed with the patient’s serum on a plastic card and the agglutination pattern is observed. Virtually all patients with Ttypanosoma brucei gambiense, and about half of those with T. b. rhodesiense, infections are detected by this test (BUSCHER et al., 19911 NOIREAU et al., 1991). which is entirelv adeauate for screening. A monoclonal antibody-based E~ISA for antigen also appearspromising (NANTULYA et al., 1992). Visceral leishmaniasis can also prove difficult to diagnose without invasive procedures and here antibody testing has proved useful both for individual diagnosis and for population screening. ELISXs have been widely used but more recently the direct agglutination test (DAT) has
498
been shown to be as sensitive and specific but easier to oerform (EL SAFI 8~E~~~s.1989). Chagas disease has always attracted the immunologists because the demonstration of antibody is virtually the only practicable way of screening for the infection on a large scale. Immunofluorescence (IFA) has been long established but disadvantages include its lack of objectivity and the labour-intensiveness. ELBA has now largely replaced IFA but still cross reactions with T. rangeli and Leishmania spp. have been a problem. This has now been overcome by the latest ELISAS(KRIEGERet al., 1992), which are based on peptide antigens exhibiting no crossreaction. These ELISASare particularly suited to blood bank screening, where the HIV and HBsAg ELISASin the same format are already well established. For the diagnosis of invasive amoebiasis, tests are carried out to detect antibody to E. histolytica. ELISA has been the method of choice (TAKEUCHI et al.. 1988). in either the microplate or dot-blot format (KAN~AR &‘VINAYAK, 1991). It is likely to be further enhanced when the recombinant antigens presently under evaluation (STANLEYet al., 1991) are introduced. The detection of E. histolytica in faeces has been more of a problem. ELISAS and dot-blot tests have had limited success but recent developments, including monoclonal antibodies which can distinguish between Dathogenic and nonpathogenic strain< offer more promise -(WONSIT et al., ~~~~:GONZALEZ-RUIZetal.. 1993). C&rdiasis is widely distributed in developing countries and the diagnosis is troublesome since it depends on microscopical examination of multiple faecal samples. ELISAS have now been developed to the point where they are sensitive and specific and they are especially well suited to enidemiological studies (GOLDIN et al., 1990). In general,‘antibody detection is of little use in giardiasis; however the demonstration of immunoglobulin M specific antibody may be useful (SULLIVANet al., 1991). It can be seen that there have recently been significant advances in the application of immunological research to the diagnostic problems with tropical parasitic diseases, and I confidently expect that, together with the new diagnostic methods develoDed bv molecular biologists (to be yeviewed in the next issue of the Transaction& these efforts will vield nractical and cost-effective benefits in the shape of’ more widely available user-friendly kits in the near future. I
Acknowledgements
I thank the Overseas Development Administration for their sup ort of the Appropriate Health Technology Programme at the E ondon School of Hygiene and Tropical Medicine.
References
Buscher, I’., Draelawts, E., Magnus, E., Vervoort, T. & Van Meirvenne, N. (1991). An experimental latex agglutination test for antibody to human African trypanosomiasis. Annales de la Societe Belge de Medecine Tropicale, 71,267-273.
Dubarry, M., Luilier, M., Malot, N., Bayard, I’., Lambin, I’., Prou, P. & Monjour, L. (1990). Enzyme immunoassays for detection of malarial antigens in human plasmas by Plasmodium falciparum monoclonal antibodies. American Journal of Tropical Medicine and Hygiene, 43,116-123.
El Safi, S. H. & Evans, D. A. (1989). A comparison of the direct agglutination test and enzyme-linked irnmunosorbent assay in the sero-diagnosis of visceral leishmaniasis in the Sudan. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,334-337.
Goldin, A. J., Apt, W., Aguilera, X., Zulantay, I., Warhurst, D. C. & Miles, M. A. (1990). Efficient diagnosis of giardiasis among nursery and primary school children in Santiago, Chile
by capture ELBA for the detection of fecal Giardia antigens. American Journal of Tropical Medicine and Hygiene, 42, 538-
545. Gonzalez-Ruiz, A., Haque, R., Rehman, T., Aguirre, A., Jaramillo, C., Castanon, G., Hall, A., Ruiz-Palacios, G., Warhurst, D. & Miles, M. A. (1993). A monoclonal antibody for distinction of invasive and non-invasive clinical isolates of Entamoeba histolytica. Journal of Clinical Microbiology, 30, 28072813. Hunt Cooke, A., Moody, A. H., Lemon, K., Chiodini, I’. L. & Horton, J. (1992). Use of the fluorochrome benzothiocarboxypurine m malaria diagnosis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86,378.
Kanwar, J. R. & Vinayak, V. K. (1991). A comparative efficacy of plate ELBA and dot ELBA to detect antiamoebic antibodies in clinical patients. Tropical and Geographical Medicine, 43, 261-265. Kerckhoven, V., Vercauteren, G., Piot, I’. & van der Groen, G. (1991). Comparative evaluation of 36 commercial assays for detecting antibodies to HIV. Bulletin of the World Health Organizati& 69,753-760. Krieger, M. A., Almeida, E., Oelemann, W., Lafaille, J. J., Pereira, J. B., Krieger, H., Carvalho, M. R. & Goldenberg, S. (1992). Use of recombinant antigens for the accurate diagnosis of Chagas’ disease.AmericanJournal of Tropical Medicine and Hygiene, 46,427-434.
Makler, M. T. & Hinrichs, D. J. (1993). Measurement of the lactic dehydrogenase activity of Plasmodium falciparum as an assessmentof parasitaemia. American Journal of Tropical Medicine and Hygiene, 48,205-210.
Mortimer, I’. I’. & Parry, J. V. (1991). Non invasive virological diagnosis: are saliva and urine substrates adeauate substitutes forblood? Reviews in Medical Virology, 1,73-78. Nantulva. V. M.. Doua. F. & Molisho. S. (1992). Diaenosis of Tryp&soma brucei gambiense sleeping ~sickness ising an antigen detection ELISA. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86,42-45.
Noireau, F., Force-Barge, I’. & Cattand, I’. (1991). Evaluation of Testryp CATT applied to samples of dried blood for the diagnosis of sleeping sickness. Bulletin of the World Health Organization, 69,607-608.
Rylatt, D. B., Kemp, B. E., Bundesen, G., John, M. A., O’Reilly, E. J., Cottis, L. E., Mites, S. J., Shaw, J. M., Dmh, D. I’., Stapleton, D. & Hillyard, C. J. (1990). A rapid whole Fp71immunoassay system. MedicalJournal ofAustralia, 152, Srivastava, I. K., Takacs, B., Caspers, I’., Certa, U., McGregor, I. A., Scaife, J. & Perrin, L. H. (1989). Recombinant polypeptides for serology of malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 83,3 17-32 1. Stanley, S. L., Jackson, T. F. H. G., Read, S. L., Calderon, J., Kunz-lenkins. C.. Gathiram. V. & Li. E. 11991). Serodiaanosis of invasive amoebiasis using a recombinant Entamoebh histolytica protein. Journal of the American Medical Association, 266,1984-1986. Sullivan, I’. B., Neale, G., Cevallos, A. M. & Farthing, M. J. G. (1991). Evaluation of specific serum anti-Giardia IgM antibody response in diagnosis of giardiasis in children. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 748-749. Takeuchi,, T., Matsuda, H., Okuzawa, E., Nozaki, T., Kobayashi, S. & Tanaka, H. (1988). Application of an ELISA to detect anti amoebic antibody in various forms of amoebic infection. JapaneseJournal of Experimental Medicine, 58, 229232. Taylor, D. W. & Voller, A. (1993). The development and validation of a simple antigen detection ELISA for Plasmodium falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 87,29-3 1.
Wonsit, R., Thammapalerd, N., Tharavanij, S., Radomyos, I’. & Bunnag, D. (1992). Enzyme-linked immunosorbent assay based on monoclonal and oolvclonal antibodies for the detection of Encamoeba histolyiica antigens in faecal specimens. Transactions of the Royal Society of Tropical Medicine and Hy-
giene,86, 166-169.